1997, 17(9):5299. Mol. Cell. Biol. 

GeiduschekG A Kassavetis, C Bardeleben, A Kumar, E Ramirez and E P promoter.and recruitment of RNA polymerase to the functions in assembly of TFIIIB-DNA complexespolymerase III transcription factor IIIB (TFIIIB): Domains of the Brf component of RNA

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MOLECULAR AND CELLULAR BIOLOGY,0270-7306/97/$04.0010

Sept. 1997, p. 5299–5306 Vol. 17, No. 9

Copyright © 1997, American Society for Microbiology

Domains of the Brf Component of RNA Polymerase III TranscriptionFactor IIIB (TFIIIB): Functions in Assembly of TFIIIB-DNA

Complexes and Recruitment of RNA Polymerase to the PromoterGEORGE A. KASSAVETIS,* CAROLYNE BARDELEBEN, ASHOK KUMAR, ENRIQUE RAMIREZ,

AND E. PETER GEIDUSCHEK

Department of Biology and Center for Molecular Genetics, University of California,San Diego, La Jolla, California 92093-0634

Received 1 May 1997/Returned for modification 12 June 1997/Accepted 18 June 1997

Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) is composed of three subunits: the TATA-bindingprotein, the TFIIB-related protein Brf, and B(. TFIIIB, which is brought to RNA polymerase III-transcribedgenes indirectly through interaction with DNA-bound TFIIIC or directly through DNA recognition by theTATA-binding protein, in turn recruits RNA polymerase III to the promoter. N-terminally deleted derivativesof Brf have been examined for their ability to interact with DNA-bound TFIIIC and with the other componentsof TFIIIB and for participation in transcription. Brf(165–596), lacking 164 N-proximal TFIIB-homologousamino acids, is competent to participate in the assembly of TFIIIB-DNA complexes and in TFIIIC-independenttranscription. Even deletion of the entire TFIIB-homologous half of the protein, as in Brf(317–596) andBrf(352–596), allows some interaction with DNA-bound TBP and with the B( component of TFIIIB to beretained. The function of Brf(165–596) in transcription has also been examined in the context of B( with smallinternal deletions. The ability of Brf with this sizable N-terminal segment deleted to function in TFIIIC-independent transcription requires segments of B( that are individually indispensable although required on aneither/or basis, in the context of complete Brf. These findings suggest a functional complementarity andreciprocity between the Brf and B( components of TFIIIB.

The yeast RNA polymerase III transcription machinery con-sists of three transcription initiation factors, TFIIIA, TFIIIB,and TFIIIC (reviewed in references 15, 24, 28, 45, and 47). Afourth component generates quantitatively more efficient tran-scription in vitro under certain conditions (13, 41), perhaps byfacilitating protein refolding or protein-protein association.The functions of TFIIIB are central to this transcription ma-chinery, because TFIIIB directly recruits RNA polymerase III(pol III) to the transcriptional start site and because TFIIIBand RNA pol III alone suffice for transcriptional initiation(26). TFIIIB has three components, TATA-binding protein(TBP), Brf, and B0 (8, 12, 22, 27, 28, 36, 40, 41); TBP and aprotein related to yeast Brf are also components of mammalianTFIIIB; it remains to be seen whether a protein analogous toyeast B0 also exists in higher eukaryotes (11, 35, 37, 42, 43).TBP and Brf, together designated B9, are readily separablefrom B0 during conventional protein purification (25), suggest-ing that these two components of TFIIIB are somewhat looselyassociated (22), although their binding can be detected byimmuno-coprecipitation (39) and by affinity chromatography(23).

TFIIIB plays a role in transcription by pol III that TFIIB andTFIID (or TFIIB and TBP) together play in pol II transcrip-tion. Like TFIIB and TBP (9, 32, 38), TFIIIB occupies up-stream sites on pol III genes that specify the locations oftranscriptional start sites. TFIIIB is brought to these sites ei-ther through the mediation of an assembly factor, TFIIIC, orby autonomous recognition of specific DNA sequence medi-ated by its TBP.

The genes encoding the subunits of yeast TFIIIB have been

identified (28, 40, 41), making the interactions of these com-ponents and their roles in initiation of transcription by pol IIIfar more accessible to analysis by biochemical and moleculargenetic methods. When Brf is made in Escherichia coli, internalinitiation of translation (34) and proteolytic processing gener-ate numerous fragments. Attachment of an affinity tag at theC-terminal end of Brf allows a subset of these products, whichconstitute a natural N-terminal deletion series, to be purified.The particular interest of such a series lies in the fact that thehomology to TFIIB is confined to the N-terminal half of Brf.Two of the N-deletion forms of Brf that are compared in theexperiments below progressively remove the N-terminal zincfinger and the first TFIIB-related repeat in the first instanceand both TFIIB homologous repeats together with a segmentthat is somewhat conserved among fungal Brf (Brf homology I)in the second case. A third deletion variant lacks all of theabove and 35 additional amino acids.

We have compared these deletion proteins with intact Brffor the ability to interact with the other components of TFIIIB,for the ability to participate in effective recruitment of pol IIIto a U6 gene promoter, and for ability to be recruited to atRNA gene by TFIIIC. Our findings suggest a functionalcomplementarity between Brf and B0, which allows Brf deletedfor a large TFIIB-homologous segment to function in the as-sembly of TFIIIB complexes on DNA and in recruitment ofRNA polymerase III.

MATERIALS AND METHODS

DNA templates. Plasmids pU6R and pU6LboxB have been described previ-ously (46). The 130-bp U6 DNA probe for the experiments shown in Fig. 5a andb was generated by PCR and labelled as described previously (30). The 59-bplabelled U6 DNA probe for the experiment shown in lanes n and o of Fig. 5a andb was generated by annealing the template and nontemplate strand oligonucle-otides shown in Fig. 6a and filling in with [a-32P]dCTP, dGTP, and exonuclease-free Klenow DNA polymerase I. U6 DNA photochemical cross-linking probes

* Corresponding author. Mailing address: University of California,San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0634. Phone: (619)534-2451. Fax: (619) 534-7073. E-mail: gkassavetis@ucsd.edu.

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239/238 and 213/212/25/23/21 were generated by annealing 9 pmol of thespecified primer to 6 pmol of the template strand (see Fig. 6a) in 10 ml of bufferA (6) and diluting twofold with a mixture providing 10 mM 5-(N9-[p-azidoben-zoyl]-3-aminoallyl)-dUTP (ABdUTP; prepared as described in reference 4), 0.2mM [a-32P]dCTP, and 2 U of exonuclease-free Klenow DNA polymerase I inbuffer A. Following synthesis for 10 min at 37°C, an aliquot of the reactionmixture was removed to assess the fidelity of incorporation. DNA synthesis wascompleted by adding all four deoxynucleoside triphosphates to 1 mM for anadditional 15 min at 37°C, and the DNA product was purified, either by gelfiltration on Sephadex G75 or by polyacrylamide gel electrophoresis and elution.The 189-bp U6 DNA probe for the experiments shown in Fig. 3 and 6d wassynthesized as a 25/23 photochemically cross-linking probe derived from a U6M13 clone (bp 2120 to 169) as described previously (6).

Proteins. TFIIIC, pol III, recombinant TBP, recombinant TBPm3, and therecombinant wild-type and internally deleted variants of B0 were purified andassayed as described previously (28, 30, 46). Recombinant B0 for the experimentshown in Fig. 2, 4, and 6b and c was purified under nondenaturing conditions asdescribed for B0(138–594) (30). The yeast-derived B0 for the experiment shownin Fig. 3 was extracted with 6 M urea from S-100 pellets and purified to thehydroxylapatite fraction described previously (28). The yeast-derived B0 for theexperiment shown in Fig. 6d was isolated from Cibacron Blue agarose-purifiedTFIIIB by chromatography on Mono S (25). Recombinant Brf and its N-termi-nally truncated variants were purified by sodium dodecyl sulfate (SDS)-polyacryl-amide gel electrophoresis as follows. Brf and its natural N-terminal deletionproducts retaining the C-terminal hexahistidine tag were purified on Ni-nitrilo-triacetic acid agarose in the presence of 7 M urea and allowed to precipitateupon dialysis, as described previously (28). The pellet was resuspended in SDSsample buffer (31), incubated for 15 min at 37°C, and loaded onto a preparative8% polyacrylamide–SDS gel. Protein bands were visualized by staining with KCl(20). Elution and renaturation followed the conditions for gel isolation of B0(27), except that the final dilution buffer also contained 500 mM NaCl and 50 mMZnSO4. Renatured Brf proteins were concentrated on Ni-nitrilotriacetic acidagarose with the nondenaturing buffer system described for B0(138–594) (30),except that the elution buffer contained 200 mM imidazole and 10 mM ZnSO4 inplace of 60 mM imidazole. The gel-isolated Brf polypeptides for the experimentsshown in Fig. 3 and 6d were separately prepared on a smaller scale, dialyzed intobuffer D100 (30), and not concentrated. The N-terminal sequence of Brfpolypeptides was determined by the University of California San Diego BiologyDepartment protein-sequencing facility. Brf(317–596) and Brf(352–596) werefound to contain Met at their N termini. The respective codons at these positions,TTG and GTG, correspond to minor translational initiation codons, indicatingthat these variants arise from gene-internal translational initiation events.

The quantities of TFIIIC are specified as femtomoles of DNA-binding activity,and the quantities of pol III are specified as femtomoles of pol III active forspecific transcription (30). Recombinant B0 (all preparations), TBP, TBPm3, andgel-isolated Brf polypeptides are specified as femtomoles of protein (estimatedrelative to a standard curve for Coomassie-blue stained bovine serum albumin onSDS-polyacrylamide gels or by the predicted extinction coefficient of the protein[wild-type B0 and TBP]). The gel-isolated Brf proteins for the experiments shownin Fig. 3 and 6d were not quantified, but their purity was confirmed on SDS-polyacrylamide gels. The yeast-derived B0 preparations were also not quantified.Wild-type and recombinant B0 (both preparations) and wild-type TBP wereestimated to be nearly 100% active for the formation of heparin-resistantTFIIIB-DNA complexes (28). Gel-isolated full-length Brf was at least 50% activein the same assay.

Assays. Protein-DNA complexes for binding, transcription, and photochemicalcross-linking assays were formed in 16 to 20 ml containing 40 mM Tris-Cl (pH8.0), 7 mM MgCl2, 3 mM dithiothreitol (or 3 to 5 mM 2-mercaptoethanol whenphotochemical cross-linking probes were used), 100 mg of bovine serum albuminper ml, 4 to 6% (vol/vol) glycerol, and 50 to 70 mM NaCl (or 25 mM potassiumacetate and 35 mM NaCl for the experiments shown in Fig. 3 and 6d). For theDNA binding assays shown in Fig. 5a and b, each sample contained 10 fmol ofa 32P-labelled, PCR-generated 130-bp U6 DNA probe, 200 ng of poly(dG-dC)-poly(dG-dC), 100 to 120 fmol of the specified B0, 100 fmol of TBP, and 100 fmolof Brf or Brf(165–596) as specified. Following complex formation for 60 min at21°C, 200 ng of poly(dA-dT)-poly(dA-dT) was added for an additional 10 minprior to native gel electrophoresis, as described previously (7). For a separate butsimilar experiment shown in lanes n and o of Fig. 5a and b, a 59-bp U6 probecontaining a TGTAAATA variant TATA box was used with 150 fmol of Brf orBrf(165–596) and 150 fmol of TBPm3 in place of wild-type TBP; the sampleswere not treated with poly(dA-dT)-poly(dA-dT) prior to electrophoresis. Sam-ples for the DNA binding reactions in Fig. 3 and 6d contained 2 fmol of a 189-bpphotoreactive U6 DNA probe, 50 ng of poly(dG-dC)-poly(dG-dC), 200 fmol ofTBP, yeast-derived B0, and the indicated Brf preparation. Following complexformation for 40 min at 21°C, 250 ng of pGEM1 (see Fig. 3) or poly(dG-dC)-poly(dG-dC) was added prior to native gel electrophoresis as described previ-ously (7), except that the gel also contained 1 mM dithiothreitol and 5 mMMgCl2 and the running buffer also contained 5 mM MgCl2.

The reaction mixtures for transcription contained 50 fmol of the specifiedplasmid DNA template, 20 fmol of pol III, 100 fmol of TBP, and 150 fmol ofwild-type B0 (see Fig. 2b and 4) or 150 fmol of TBP and 100 to 120 fmol of thespecified B0 (see Fig. 5c and d), with the quantities of Brf and TFIIIC specified

in the figure legends. Transcription complexes were formed for 60 min at 21°C,followed by 30 min of multiple-round transcription, as described previously (30).

For the photochemical cross-linking experiments shown in Fig. 6b and c, thesamples contained 20 fmol of the specified 59-bp U6 DNA probe, 100 ng ofpoly(dG-dC)-poly(dG-dC), 75 fmol of B0, 200 fmol of TBPm3, and 200 fmol ofthe specified Brf preparation. Complexes were formed for 60 min at 21°C. UVirradiation and nuclease digestion prior to electrophoresis on 10% polyacryl-amide–SDS gels was carried out as described previously (6).

RESULTS

Purification of intact and N-truncated Brf. When they areoverexpressed in E. coli, intact Brf and several larger Brf frag-ments (Fig. 1a) are relatively insoluble and more readily puri-fied under denaturing conditions. Purification of C-terminallyHis-tagged intact and N-terminally deleted Brf was completedby separation on SDS-polyacrylamide gels and renaturation, asdescribed in Materials and Methods. The purity of the finalmaterials is shown in Fig. 1b.

Brf(165–596). Brf(165–596), which lacks the N-terminal Znfinger and the first TFIIB-related repeat segment, is able toreconstitute a variant TFIIIB that functions in TFIIIC-inde-pendent pol III transcription of the yeast SNR6 (U6 RNA)gene. SNR6 has a very strong TATA box binding site for TBP(TATAAATA) to which wild-type TFIIIB binds in either ori-entation, generating bidirectional transcription (46). We exam-ined transcription on the U6 gene-derived construct pU6R(Fig. 2a), which allows the orientation of TFIIIB reconstitutedwith N-truncated Brf to be assessed (46), and found a consis-tent small difference in the orientation of TFIIIB and TFIIIB-[Brf(165–596)] on the TATA box of this construct, reflected ina relative disfavoring of rightward transcription when TFIIIB isassembled with Brf(165–596) instead of full-length Brf.

It is surprising to find Brf(165–596) competent for TFIIIC-

FIG. 1. Brf. (a) Significant features of the amino acid sequence of Brf includethe TFIIB-homologous N-terminal putative Zn-binding domain and sequencerepeat, as well as three segments of conserved sequence among fungal Brfproteins. The N-terminally deleted and C-terminally His6-tagged Brf proteinsused in this work are also indicated. (b) Purity of Brf(165–596), Brf(317–596),and Brf(352–596). Coomassie-stained SDS-polyacrylamide gels of these proteinsare shown. Molecular mass markers, in kilodaltons, are indicated at the side.

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independent transcription, although it was clearly less activethan was intact Brf (Fig. 2b). The titer determinations shown inFig. 2b suggest that the observed differences of transcript yieldbetween Brf and Brf(165–596) arise from differences in abso-lute rather than specific activity of these two protein prepara-tions, since the ratio of leftward transcription for Brf relative toBrf(165–596) was approximately the same at nearly limitingand plateau concentrations of these two proteins. (Excess Brfappeared to be somewhat inhibitory for rightward transcrip-tion.)

Consistent with its ability to generate transcription, Brf(165–596) also formed a complex with U6 DNA, TBP, and B0 thatwas stable to electrophoresis on native polyacrylamide gels,where it had a slightly greater mobility than did the intactTFIIIB-DNA complex (Fig. 3a, compare lane e with lane a).The complex was, however, much more sensitive to dissocia-tion by heparin (lanes f to h) and by electrolyte (Fig. 3b) thanwas the DNA complex containing intact Brf.

The interaction of Brf(165–596) with TFIIIC is, however,defective. An incisive way of examining this interaction is pro-vided by the experiment presented in Fig. 4. The U6 gene-derived plasmid U6Lbox B (Fig. 2a) is useful for examining the

effect of TFIIIC on the orientation of TFIIIB at the U6 geneTATA box. The rightward transcription unit of this constructhas the U6 gene’s weak box A and an inserted box B separatedby an optimal 49 bp (1). In the presence of prebound TFIIIC,TFIIIB binds to the upstream SNR6 TATA box almost exclu-sively in the direction that yields rightward transcription (r-U6)of the constructed gene. Once TFIIIB has bound to the TATAbox, it is not reoriented by subsequently added TFIIIC (46).pU6Lbox B generates two additional transcripts (r-d and l-d)mediated by a second TATA box (d; Fig. 2a) that should not beaffected by the addition of TFIIIC. A titration with TFIIICshowed transcription directed by complete TFIIIB respondingin the expected manner so as to favor the production of right-ward (r-U6) transcripts (Fig. 4). In contrast, TFIIIB reconsti-tuted with Brf(165–596) did not change its orientation on theTATA box in response to prebound TFIIIC.

Consistent with the preceding findings, we did not detecttranscription of the SUP4 gene above a very low background ofTFIIIC-independent transcription (primarily initiating at bp18 [data not shown]) when TFIIIB was reconstituted withBrf(165–596). The lack of TFIIIC-dependent transcription re-flected the inability of Brf(165–596) to generate B9(Brf1TBP)-TFIIIC-SUP4 tDNA and TFIIIB-TFIIIC-SUP4 tDNA com-plexes in electrophoretic mobility shift assays (data not shown).

Functional reciprocity of Brf and B(. A functional dissectionof the B0 component of TFIIIB has been undertaken recently(30). The analysis reveals B0 as a scaffold for TFIIIB with

FIG. 2. Brf(165–596) is transcriptionally competent. (a) The pol III tran-scription units of constructs pU6R and pU6Lbox B are shown. DNA segmentsidentical to the S. cerevisiae U6 RNA gene (SNR6) are indicated:h<<<< , leftwardorientation;h>>>> , rightward orientation. Each construct has a secondary TATAbox, d, specifying a second pair of divergent transcripts. The SNR6 and d TATAboxes are identical (TATAAATA). r-d, r-U6, l-U6, and l-d designate the 173- to179-, 112- to 118-, 98- to 104-, and 38- to 44-nucleotide transcripts, respectively(the heterogeneity in length resulting from termination within the last 6 Tresidues of a T10 termination sequence). (b) Transcription of pU6R as a functionof the concentration of Brf proteins. E, F, rightward transcripts (specified by theSNR6 TATA box) with Brf and Brf(165–596), respectively; h, ■, leftward tran-scripts (specified by the SNR6 TATA box) with Brf and Brf(165–596), respec-tively. An example of the primary data, showing transcripts produced with 50fmol of Brf and Brf(165–596), is presented at the right of the graph.

FIG. 3. Formation of heparin-sensitive and salt-sensitive DNA complexes atthe SNR6 TATA box by TFIIIB reconstituted with Brf(165–596). (a) DNA, Brf(lanes a to d), Brf(165–596) (lanes e to h), and B0 were incubated for 40 min, andnonspecific competitor DNA was added for 10 min. Heparin, at the concentra-tions noted above each lane, was added for another 5 min, and then the sampleswere loaded for gel electrophoresis. The locations of TFIIIB-DNA complexesand free DNA on the gel are indicated at the side. (b) Samples were assembledas specified above, but NaCl at the final concentration shown on the abscissa wasadded for 10 min, in place of heparin, before the samples were loaded for gelelectrophoresis. Residual TFIIIB-DNA complexes (ordinate) were quantifiedwith a PhosphorImager. E, TFIIIB-DNA complex; ■, TFIIIB[Brf(165–596)]-DNA complex.

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multiple roles in the assembly of promoter complexes andinitiation of transcription. Extensive deletions in B0 are com-patible with transcription in vitro. Two domains near aminoacids 290 and 430 (regions II and I, respectively, defined byscanning with 12- to 20-amino-acid deletions) are essential forTFIIIC-dependent transcription but are required only on aneither/or basis for TFIIIC-independent transcription of theSNR6 gene.

Since Brf(165–596) functions in U6 transcription and par-ticipates in forming TFIIIB complexes on the SNR6 gene, weanalyzed it in the context of the collection of B0 internal de-letion mutants. The results were clear-cut and striking (Fig. 5aand b): when the N-proximal 164 amino acids of Brf wereremoved, the demands on B0 became reciprocally more strin-gent. Domains I and II of B0 once again came to prominencebut in a new way: they were now separately required for for-mation of TFIIIB-DNA complexes with the SNR6 gene.

In region II, B0D272–292, which forms heparin-sensitivetranscription complexes with intact Brf on the U6 gene (30),failed to form DNA complexes or support transcription on thesame gene with Brf(165–596) (Fig. 5b and d, lanes d). B0 withthe adjacent amino acids 291 to 310 or 312 to 325 deleted wasfunctional for TFIIIC-dependent as well as TFIIIC-indepen-dent transcription and formed heparin-resistant TFIIIB-DNAcomplexes with intact Brf (30). In contrast, B0D291–310 failedto form TFIIIB-DNA complexes with Brf(165–596) (Fig. 5b,lane e) and did not support SNR6 transcription (Fig. 5d, lanee), while B0D312–325 formed TFIIIB-DNA complexes poorly

(Fig. 5b, lane o) and generated an aberrant transcription pat-tern on pU6Lbox B (Fig. 5d, lane f), with rightward transcrip-tion specified by the SNR6 TATA box (r-U6) preferentially lost(Fig. 5e). Thus, the N-terminally truncated Brf(165–596) ex-posed the functional relevance of an extended B0 domain II.

In region I of B0, deletions of amino acids 409 to 421, 424 to438, and 438 to 449 generated proteins that participated informing TFIIIB complexes on the SNR6 gene with intact Brfand were competent for transcription (Fig. 5a, lanes k to m,and 5c, lanes l to n), but no complexes were detected withBrf(165–596) (Fig. 5b, lanes k to m) and there was no tran-scription (Fig. 5d, lanes l to n). B0 with the adjacent aminoacids 355 to 372, 372 to 387, or 388 to 409 deleted is functionalfor TFIIIC-independent and TFIIIC-requiring transcription inconjunction with intact Brf (30) (Fig. 5c), but in the context ofBrf(165–596) these B0 deletions generated a novel phenotype:formation of TFIIIB-DNA complexes (Fig. 5b, lanes h to j)that were transcriptionally inactive (Fig. 5d, lanes i to k). In-activity could be due to an inability to recruit pol III or tofailure at some subsequent step (promoter clearance, for ex-ample).

The preceding experiments demonstrate a functional reci-procity between Brf and B0: removing a segment of one ofthese proteins makes the demands on its partner more strin-gent. In the context of full-length B0, it is possible to remove alarge segment of Brf that is homologous to TFIIB and stillretain competence for TFIIIC-independent transcription.Since the homology between TFIIB and Brf is likely to be

FIG. 4. Brf(165–596) does not interact with TFIIIC: a transcription experiment with pU6Lbox B. Buffer or TFIIIC in the amounts specified at the top of panel a(13 5 9 fmol) was incubated with pU6Lbox B for 7 min at 21°C. A protein mixture containing a fixed quantity of pol III, TBP, B0, and 50 fmol of Brf or Brf(165–596)was added for an additional 60 min prior to 30 min of multiple-round transcription. (a) Transcripts specified by the SNR6 and d TATA boxes are identified at the right.The labelled DNA sample recovery standard (r.m.) is also identified. (b) Quantitative analysis of the results in panel a.

FIG. 5. The role of B0 in forming TFIIIB-DNA complexes with Brf(165–596) and in initiation of transcription: a survey of internal deletions in B0. (a and b)Complexes of 32P-labelled SNR6 DNA with TFIIIB reconstituted with Brf (a) or Brf(165–596) (b), and the indicated internally deleted B0 proteins were analyzed byelectrophoresis on native gels. Deletions extending into domains I and II of B0 (see the text) are indicated (DI and DII, respectively). The reaction mixtures analyzedin lanes n and o of panels a and b were from a separate experiment (see Materials and Methods). TFIIIB-DNA and B9(Brf1TBP)-DNA complexes are identified atthe left of the panels. The weak complex migrating above the TFIIIB-DNA complex in panel b, when less than 50% of the free DNA probe remains, probably representsa second TFIIIB or B9 complex bound to imperfect, TATA-like sequences. We have observed complexes of this DNA with multiple molecules of TBP (data not shown).(c and d) The same series of B0 deletion proteins was tested for TFIIIC-independent transcription with 100 fmol of Brf (c) and Brf(165–596) (d). Rightward and leftwardtranscripts specified by the SNR6 and d TATA boxes (Fig. 2a) are indicated at the right and quantified in panel e.

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reflected in structural similarity (38), it is appropriate to con-sider this finding in the context of the structure of the TFIIB-TBP-TATA box complex. The N terminus of TFIIB probablyextends along DNA between the TATA box and the transcrip-tional start site (32, 38). Diverse evidence implicates this es-sential N-terminal segment in interaction with pol II-TFIIF(19, 21). The N-proximal first repeat of human TFIIB (corre-sponding to amino acids 140 to 210 of yeast TFIIB) interacts atmultiple points with the C-proximal domain (repeat 2) of TBP(38). That the corresponding segments of Brf can be removedwithout totally eliminating transcription by pol III or TFIIIBassembly on DNA emphasizes the importance of B0 and sug-gests that B0 can play a direct role in recruiting pol III.

An unplanned feature of the design of the SNR6 gene inplasmid pU6Lbox B offered an opportunity to observe effectsof mutations in B0 and Brf on promoter selection. As notedabove, the leftward transcription unit in pU6Lbox B contains asecond TATA box (d in Fig. 2a) that generates its own pair ofdivergent transcripts. The two TATA boxes in pU6Lbox B areidentical, TATAAATA(A), and have the same orientation, buttheir flanking sequences and the sequences immediately sur-rounding the transcriptional start sites differ. Thus, the factthat the relative proportions of the d transcripts and the pU6Ltranscripts differ for different combinations of B0 and Brf pro-teins (Fig. 5e) signifies that Brf and B0 can influence promoterselection. In the context of full-length Brf, B0D409–421,

B0D424–438, and B0D438–449 are fully functional for SNR6transcription (30) but clearly yielded less rightward transcrip-tion from the d promoter. Conversely, rightward transcriptionfrom the d promoter was generally higher (relative, for exam-ple, to leftward transcription from the SNR6 promoter) in thecontext of Brf(165–596) than in the context of full-length Brf.We discuss the implications of these findings below.

Brf(317–596) and Brf(352–596). Brf(317–596) lacks all ofthe TFIIB-homologous N-proximal half of Brf yet retains someability to bring TBP and B0 into a common complex with DNA.Formation of this complex, which was analyzed primarily byphotochemical cross-linking, clearly showed that Brf(317–596)binds to TBP-DNA complexes, just as intact Brf and Brf(165–596) do, and that Brf(317–596) is cross-linked to DNA withonly slightly lower efficiency (Fig. 6b and c, lanes d to f). DNAbinding required TBP (lanes a to c). The Brf(317–596)-TBP-DNA complex also bound B0, just as the complexes with intactBrf and Brf(165–596) did (lanes g to i). However, the efficiencyof cross-linking B0 to DNA, both upstream and downstream ofthe TATA box, was much lower in complexes formed with

FIG. 6. Brf(317–596) and Brf(352–596) can be brought into SNR6 DNAcomplexes with TBP and B0. Analysis by photochemical cross-linking is shown.(a) Sequence of the SNR6 DNA segment that was used to analyze proteinbinding by photochemical cross-linking. The indicated primers (underlined) wereused to insert photoactive ABdUMP (u) and [32P]dCMP (c) by primer extension,as shown. Synthesis of these strands was completed with unlabelled nucleotides.(b and c) Photochemical cross-linking at the upstream (b) (probe 239/238) anddownstream (c) (probe 213/212/25/23/21) sides of the TATA box. Samplescontaining TBP, Brf, Brf(165–596) or Brf(317–596), and B0, as indicated aboveeach lane, were incubated with DNA, irradiated, digested with nucleases, andresolved on SDS–10% polyacrylamide gels. Samples for lanes j to l of each panelwere challenged with 200 mg of heparin per ml for ;10 min before beingirradiated. DNA-linked proteins are identified at the sides. (d) Electrophoreticmobility shift analysis. A U6 DNA probe was incubated with TBP, B0, andBrf(352–596), as indicated above each lane for 40 min, and then nonspecificcompetitor DNA was added for another 10 min before the samples were loadedfor native gel electrophoresis. TFIIIB-DNA, B9-DNA, and TBP-DNA complexesare identified at the right.

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Brf(317–596) (Fig. 6b and c, lanes i) than in those with thelarger Brfs (lanes g and h), although it was clearly dependenton Brf(317–596) (compare lanes m and n with lane i). Only theTFIIIB-DNA complex formed with full-length Brf was stableto challenge with heparin (Fig. 6b and c, lanes j to l).

These results indicate that the C-terminal half of Brf, on itsown, retains a capacity for interaction with DNA-bound TBPand with B0. The complex with B0 is held together weakly, butit is specific, in the sense that it requires TBP as its DNAanchor. Even Brf(352–596) retained the ability to bind to TBP-DNA and secure entry of B0 into a DNA complex (Fig. 6d,lanes e and f). These interactions were specific, in the sensethat B0 did not bind directly to DNA (lane a) or to a TBP-DNAcomplex (lane b) and that Brf(352–596) bound to DNA in thepresence but not the absence of TBP (lanes b and e).

DISCUSSION

The interaction between TFIIB and TBP is mediated pri-marily by the carboxy-terminal end of the first imperfect repeatof TFIIB (19, 38, 48). The corresponding region in Brf isclearly not a major determinant in Brf-TBP interactions. It hasbeen shown (10, 29) that the C-proximal half of Brf binds toTBP, and we see a manifestation of this in the formation ofDNA complexes (Fig. 6). Our experiments also show that Brflacking the N-proximal TFIIB-homologous repeat can recruitB0. Even removal of the entire TFIIB homology region and thefungal homology region I of Brf does not entirely eliminate theability to incorporate B0 into a TFIIIB DNA complex. On theother hand, TFIIIC interaction is seriously compromised in theBrf(165–596) mutant. This is consistent with the prior work ofChaussivert et al. (10), who found that the two TFIIB-relatedrepeats (but not the Zn finger) of Brf interact with the t120subunit of TFIIIC. The effect of N- and C-terminal deletionvariants of Brf on yeast viability have been examined by Col-bert and Hahn (12), who deleted the first 40 amino acids of Brf(the Zn finger domain), generating a lethal phenotype. Al-though this region of Brf appears not to be a major determi-nant for interaction with pol III, TFIIIC, B0, or TBP (10, 29)(see above), it may nevertheless contribute to the stability ofthe TFIIIB-DNA complex (since removal of the first 164amino acids of Brf generates TFIIIB-DNA complexes that areheparin sensitive), with a stable TFIIIB-DNA complex beingessential for viability.

The N-terminal ;90-amino-acid domain of TFIIB, whichpoints from the TATA box toward the transcriptional start, isimportant for TFIIF-mediated assembly of pol II into the TBP-TFIIB-DNA complex (19, 38, 48). However, the direct inter-face between TFIIB and pol II appears to lie at the carboxy-terminal end of the second imperfect TFIIB repeat (19). Thefact that Brf(165–596), which retains this region, functions forpol III assembly and transcription while Brf(317–596), whichlacks this region, is inactive might likewise reflect an interac-tion with pol III within or just C-terminal of the second repeatin Brf. However, Khoo et al. (29) found the first repeat of Brf(but not the second repeat) interacting with pol III through its34-kDa subunit. Our finding therefore provides a strong argu-ment for an additional site of interaction between pol III andTFIIIB and for an active role of B0 in pol III recruitment.

There is a functional complementarity between Brf and B0:the ability of Brf(165–596) to form a TFIIIB-DNA complex onthe SNR6 gene and to function in TFIIIC-independent tran-scription is dependent on segments of B0 that are dispensablein the context of complete Brf (Fig. 5). It is interesting tocompare the regions of B0 that specifically become indispens-able under these circumstances with the results of a prior

exploration of the surface topography of B0 by hydroxyl radicalfootprinting (Fig. 7). The deletions that make B0 unable toform TFIIIB-DNA complexes and, as a consequence, unableto support transcription by pol III fall in region I (deletionscovering amino acids 409 to 449) and region II (deletionscovering amino acids 272 to 310, with a strong inhibitory effectdue to deleting amino acids 312 to 325). The formation ofTFIIIB-DNA complexes protects B0 regions I and II fromcleavage by zOH, presumably by burying them. Certain com-binations of deletions in regions I and II of B0 inactivateTFIIIC-independent transcription (30).

Deletions within the amino acid 355 to 409 segment of B0are particularly interesting because they generate B0 proteinsthat enter into transcriptionally inactive TFIIIB complexeswith Brf(165–596) (Fig. 5b and d). This segment of B0 is espe-cially reactive to zOH in the free protein and in TFIIIB-DNAcomplexes, as though it were fully exposed at the surface. Thetranscriptional inactivity of the TFIIIB-DNA complex assem-bled with Brf(165–596) and B0D355–372 strongly implicatesthe outward-facing amino acid 355 to 372 segment of B0 ininteraction with pol III. Prior analysis of the alignment of B0with DNA by photochemical cross-linking showed that a seg-ment of this protein lies close to DNA between the TATA boxand the transcriptional start site and therefore places it in thevicinity of the space in which a B0-pol III interaction wouldhave to take place (2, 3, 5). That TFIIIB reconstituted withfull-length Brf and the same internally deleted B0 is transcrip-tionally functional (30) (Fig. 5c) implies the existence of asecond TFIIIB-pol III interaction, presumably the alreadymentioned interaction between Brf and the C34 subunit of polIII (44) involving the N-proximal 164 amino acids of Brf (29).

It is perhaps surprising to see mutations in Brf and B0 gen-erating direct effects on promoter selection in TFIIIC-inde-pendent transcription. These effects presumably are directlyattributable to changes in the distribution of TFIIIB betweenleft- and right-facing orientations at the d and U6 TATAboxes. The sequences of upstream segments of pol III-tran-scribed genes vary greatly (16, 45), and large-scale substitu-tions of sequence upstream of the SNR6 transcriptional startsites are readily tolerated in vivo (14, 17). We suspect that theeffects observed by us are related to very sharp DNA bendingin the TFIIIB-DNA complex (33) and sequence-dependentdeformability of DNA. It seems likely that DNA is bent byTFIIIB at additional sites outside the TATA box (18). If thatis the case, different sequences at those additional sites shouldmake unequal contributions to the energetics of complex for-mation. These differential contributions might change, andalso become more significant, as interactions within TFIIIB areweakened by mutations introduced into its subunits. We sug-gest that these changes may generate the observed effects ofmutations in Brf and B0 on promoter utilization.

FIG. 7. Internal domains of B0 that are essential in the context of Brf(165–596) for formation of TFIIIB-DNA complexes and for TFIIIC-independenttranscription. The locations of B0 internal deletions that eliminate the ability toform TFIIIB-DNA complexes with Brf(165–596) (■) or allow the formationof transcriptionally inactive TFIIIB-DNA complexes (h) are shown. Thepreviously identified domains I and II of B0 are also shown, with the dots at thesides indicating that the full extent of these domains is not known. A region ofB0 that is highly exposed at the surface of TFIIIB-DNA complexes is marked(zzXXXzz).

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ACKNOWLEDGMENTS

We thank Cynthia J. Brent for assistance and Claudio A. P. Joazeirofor helpful comments.

C.B. was a trainee supported by a training grant from the NIGMS.Our research was supported by a grant from the NIGMS.

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